Aug 5, 1991 - Gene expression of human cytomegalovirus (HCMV), a member of the beta subgroup of herpesviruses, is regulated in a cascade (10, 11, 21, ...
JOURNAL OF VIROLOGY, Nov. 1991, p. 6301-6306 0022-538X/91/116301-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 65, No. 11
An Inducible Promoter Mediates Abundant Expression from the Immediate-Early 2 Gene Region of Human Cytomegalovirus at Late Times after Infection ELKE PUCHTLER AND THOMAS STAMMINGER* Institut fur Klinische und Molekulare Virologie der Universitat Erlangen-Nurnberg, Loschgestrasse 7, 8520 Erlangen, Germany
Received 22 May 1991/Accepted
5
August 1991
An abundant late transcript of 1.5 kb originates from the immediate-early 2 (IE-2) gene region of human cytomegalovirus (HCMV) at late times after infection. The transcriptional start site of this RNA was precisely mapped, and the putative promoter region was cloned in front of the CAT gene as reporter. This region, which comprises 78 nucleotides of IE-2 sequence upstream of the determined cap site, was strongly activated by viral superinfection at late times in the replicative cycle. As shown by RNase protection analyses, the authentic transcription start is used. No activation of this late promoter was observed after cotransfection with an expression plasmid containing the HCMV IE-1 and -2 gene region. This result suggests that, compared with early and early late promoters of HCMV, different or additional viral functions are required for the activation of true late promoters.
Gene expression of human cytomegalovirus (HCMV), a member of the beta subgroup of herpesviruses, is regulated in a cascade (10, 11, 21, 36, 37). Three broad phases of gene expression can be distinguished. Immediate-early (IE) genes are expressed in the absence of viral protein synthesis. Their gene products are involved in the switch to the early phase (20, 30). Late genes have been classified into two groups: early-late genes (also called -yl genes), which are readily detectable prior to and whose expression increases after viral DNA replication; and (true) late genes (also called -y2 genes), which are only detectable after viral DNA replication. Few true late genes have been identified within HCMV (22, 25, 29), and the mechanisms involved in their activation remain to be determined. One gene locus within HCMV, referred to as the IE-1 and -2 gene region (UL 123-122 [7]), is abundantly transcribed at IE times of infection (18, 33, 38). A strong enhancer element is located immediately upstream of the IE-1 cap site (5, 34). RNAs derived from IE-1 and -2 show a complex splicing pattern (1, 31, 32). Since the first three exons of IE-1 are differentially spliced onto exons of IE-2 (32), it can be shown that the IE-1 enhancer drives expression of both IE-1 and -2 RNAs. For HCMV strain Towne it has been reported that the IE-2 gene region is also heavily transcribed at late times after infection (29, 32). In particular, an abundant late RNA of 1.5 kb originating from within IE-2 in HCMV strain Towne was mapped (31). Using the prototype HCMV strain AD169, we confirmed the late expression and the transcriptional start site of the 1.5-kb IE-2 RNA. We demonstrate that a trans-activatable promoter region is composed within a fragment of 78 nucleotides (nt) upstream of the mapped cap site. Although proteins as expressed in transient assays from IE-1 and -2 have been shown to trans activate both homologous and heterologous promoters (20, 24, 28), the identified promoter region could not be activated in cotransfection experiments using the major IE-1 and -2 gene region; how-
*
ever, the promoter region strongly responded to viral superinfection. Northern (RNA) blot experiments were performed to investigate expression of RNAs from the IE-2 gene region of HCMV during the replicative cycle. Human foreskin fibroblasts (HFFs) were infected with HCMV strain AD169 at a multiplicity of infection of 10, and total cellular RNA was harvested at indicated times after infection by the method of Chomczynski and Sacchi (9). After electrophoretic separation of RNA on formaldehyde gels and transfer to nitrocellulose filters as previously described (27), hybridizations were performed by using the SmaI-XhoI fragment of the IE-2 gene region, labeled by nick translation, as a probe (Fig. 1A). At IE times the major signal observed with the IE-2specific probe was at a size of 2.2 kb (Fig. 1B, lane 1). A strong reduction in abundance of this 2.2-kb RNA species was found during early times and at late times of infection (Fig. 1B, lanes 2 to 4). At late times additional signals appeared when the IE-2-specific probe was used; the most prominent signal migrated at a size of 1.5 kb (Fig. 1B, lane 4). To test whether expression of this late transcript is dependent on prior viral DNA replication, RNA was harvested at late times after infection in the presence of a 200-,ug concentration of phosphonoformic acid, which is known to be a potent inhibitor of herpesvirus DNA polymerases (35). After hybridization with the IE-2-specific probe, no signal could be detected at 1.5 kb (Fig. 1C, lane 2). Thus, the pattern of transcripts originating from IE-2 changes during the time course of infection. Whereas at IE times an RNA of 2.2 kb dominates, this region is also abundantly transcribed at late times after infection. When hybridizations were performed by using probes that are specific for exon 1 or exon 4 of IE-1, the 1.5-kb RNA could not be detected (27), suggesting that expression of this RNA is not driven by the major IE enhancer/promoter. Primer extension analyses to map the transcriptional start site of the late 1.5-kb RNA were then performed. A 30-mer oligonucleotide primer that was complementary to nt 3218 to 3247 relative to the IE-1 cap site and maps to a position 399 nt downstream of the IE-1 polyadenylation signal was syn-
Corresponding author. 6301
6302
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